Preparation method of Cryptococcus neoformans capsular polysaccharide GXM as well as GXM antigen immunoassay kit and application thereof

ABSTRACT

The present invention discloses a preparation method of Cryptococcus neoformans capsular polysaccharide GXM as well as a GXM antigen immunoassay kit and an application thereof. The preparation method of the Cryptococcus neoformans capsular polysaccharide GXM effectively avoids the use of a toxic chemical reagent, ensures safety of operators, and also avoids environmental pollution, has high specificity, and can prepare a high-purity Cryptococcus neoformans capsular polysaccharide while simplifying a preparation process. The GXM antigen immunoassay kit adopts a competition method, has good sensitivity, specificity, repeatability and stability, has high recovery rate of a target compound and may provide more accurate and reliable inspection results. The kit is simple and feasible in use and operation, rapid and sensitive in detection and low in price and provides an effective tool for clinical detection of GXM.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/CN2017/074700 with a filing date of Feb. 24, 2017, designatingthe United States, now pending, and further claims priority to ChinesePatent Application No. 201610112315.4 with a filing date of Feb. 29,2016, No. 201610112389.8 with a filing date of Feb. 29, 2016, and No.201610112390.0 with a filing date of Feb. 29, 2016. The content of theaforementioned applications, including any intervening amendmentsthereto, are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the technical field of immunoassay, andparticularly relates to a preparation method of Cryptococcus neoformanscapsular polysaccharide GXM (glucuronoxylomannan), a polyclonal antibodyand a preparation method of the polyclonal antibody with respect to theGXM antigen, and an immunoassay kit as well as a preparation method andan application thereof with respect to the GXM antigen.

BACKGROUND OF THE INVENTION

Cryptococcus neoformans is an important conditioned pathogen, and ofteninfects patients with low immunocompetence or immunodeficiency, causingdeep fungal infection with a principle symptom of central nervous systeminfection. The deep fungal infection may cause an extremely high casefatality rate. Large series of epidemiologic study performed by Centerfor Disease Control and Prevention (CDC) in 1992-1993 showed that anannual incidence rate of the deep fungal infection is 178.3/million,wherein the annual incidence rate of cryptococcosis is 65.5/million,accounting for about 36.7%.

In recent years, because of long-term wide applications ofbroad-spectrum antibacterial drugs, adrenocortical hormone, tumorchemotherapy, radiation oncology and immunosuppressors after organtransplantation as well as the epidemic of AIDS, the cryptococcosis isobviously increased. Cryptococcus neoformans meningitis (hereinafterreferred to as cryptococcal meningoencephalitis) is the most commoncentral nervous system disease caused by the Cryptococcus neoformans,and accounts for about 80% of the cryptococcosis. Due to complicatedclinical manifestations and atypical symptoms, the cryptococcalmeningoencephalitis is difficult to be diagnosed. About 80% of patientssuffering from the cryptococcal meningoencephalitis may be misdiagnosedwith tubercular meningitis.

Primary causal factors of the Cryptococcus neoformans are capsule,melanin, growth capability thereof in a host temperature environment,etc. The capsule is one of the main determination factors that influencetoxicity of the Cryptococcus neoformans. A main component of the capsuleis capsular polysaccharide and has the effects of inhibitingphagocytosis and activating complement, and the component with thehighest content is glucuronoxylomannan (GXM). In an early diagnosismethod of the cryptococcus neoformans meningitis, Wang et al. detect aCryptococcus neoformans capsular polysaccharide antigen by adopting alatex agglutination test and evaluate diagnostic sensitivity andspecificity by fungal culture and direct microscopic examinationmethods. Results show that the sensitivities of the latex agglutinationtest, the fungal culture and the direct microscopic examination arerespectively 91.1%, 69.6% and 73.2%, and the specificities arerespectively 96.0%, 100% and 100%. Thus, it is believed that, the latexagglutination test for detecting the Cryptococcus neoformans capsularpolysaccharide antigen can serve as the early diagnosis method of thecryptococcus neoformans meningitis. The method for performingcryptococcus latex agglutination test on the cryptococcus neoformansmeningitis has high specificity in diagnosis of the cryptococcusneoformans meningitis, so that an early diagnosis rate is increased(Wang H, Yuan X, Zhang L. Latex agglutination: Diagnose the earlycryptococcus neoformans test of capsular polysaccharide antigen.Pakistan journal of pharmaceutical sciences, 2015, 28(1 Suppl):307-311). Therefore, the Cryptococcus neoformans capsular polysaccharideis very important to early diagnosis of the cryptococcus neoformansmeningitis.

Kozel et al. perform the following steps: culturing the cryptococcusneoformans in a culture tank, performing autoclaved sterilization on aculture solution, centrifuging to remove thalli, filtering thesupernatant with a filter membrane of 0.45 um, performingultrafiltration concentration, adding sodium acetate and glacial aceticacid into the concentrated solution, and precipitating a coarse capsularpolysaccharide with ethanol; repeatedly extracting withchloroform/n-butyl alcohol (v:v=5:1), removing protein, precipitatingthe capsular polysaccharide with ethanol, centrifuging and collectingthe precipitate, dissolving the precipitate in deionized water,dialyzing and freeze-drying, thereby obtaining refined capsularpolysaccharide. (Kozel T R, Cazin J. Nonencapsulated variant ofCryptococcus neoformans I. Virulence studies and characterization ofsoluble polysaccharide. Infection and immunity, 1971, 3(2): 287-294).Lots of chloroform should be used in the above method. The chloroform isa toxic and harmful dangerous chemical, has carcinogenic risk and alsohas high requirements for operating conditions, protection of operators,sewage treatment and the like.

Bryan et al. perform the following steps: washing cryptococcusneoformans cells with distilled water for 3 times, centrifuging andcollecting the cells, resuspending the collected wet cells with 15 mL ofdimethyl sulfoxide (DMSO) and incubating for 30 minutes; precipitatingand separating the cells by centrifuging, dialyzing DMSO supernatant for12 hours, exchanging water every 2 hours, fully dialyzing the obtainedsample with distilled water or 1 mM EDTA for 3 days, and performingfreeze drying on a polysaccharide solution obtained by dialyzing,thereby obtaining the capsular polysaccharide (Bryan R A, Zaragoza O,Zhang T, et al. Radiological studies reveal radial differences in thearchitecture of the polysaccharide capsule of Cryptococcus neoformans.Eukaryotic Cell, 2005, 4(2): 465-475). The above method is long inpreparation cycle, and the yield of the product is low.

Maxson et al. perform the following steps: centrifuging the culturefluid of the cryptococcus cells, separating a cell precipitate,concentrating the obtained supernatant by about 20 times by using apolyether sulfone ultrafiltration plate, continuously stirring in theconcentrating process, discharging a fluid phase after a sticky film isformed on the filtration plate, recovering binding material by using acell scraper, and finally performing freeze drying, thereby obtainingthe capsular polysaccharide (Maxson M E, Dadachova E, Casadevall A, etal. Radial mass density, charge, and epitope distribution in theCryptococcus neoformans capsule. Eukaryotic cell, 2007, 6(1): 95-109).The above method is simple in operation and short in preparation cycle,but the yield of the product is also low.

In addition, Frases et al. find in experiments that, extracellularpolysaccharide and the capsular polysaccharide of the cryptococcus arestructurally different, and also find in experiments that, by comparingpolysaccharide prepared by precipitating through cetyl trimethylammonium bromide with polysaccharide prepared by concentration andultrafiltration (see an extraction method in Maxson M E, Dadachova E,Casadevall A, et al. Radial mass density, charge, and epitopedistribution in the Cryptococcus neoformans capsule. Eukaryotic cell,2007, 6(1): 95-109), the mass is greater, and conformations of thepolysaccharides are different. Then, it is judged that, a common methodfor extracting the Cryptococcus neoformans capsular polysaccharide mayobviously influence the structure and antigenicity of the product(Frases S, Nimrichter L, Viana N B, et al. Cryptococcus neoformanscapsular polysaccharide and exopolysaccharide fractions manifestphysical, chemical, and antigenic differences. Eukaryotic cell, 2008,7(2): 319-327).

At present, there is an urgent need to develop a preparation method ofthe Cryptococcus neoformans capsular polysaccharide with convenience,high efficiency, low cost and high specificity in the field, and thepreparation method shall effectively avoid the use of any toxic andharmful chemical reagent. The present invention overcomes the defects inthe prior art, provides a preparation method of Cryptococcus neoformanscapsular polysaccharide GXM, the prepared GXM, and a detection kitprepared by utilizing the GXM. The prepared GXM has high purity andspecificity. The detection kit prepared by utilizing the antigen hashigh detection sensitivity.

SUMMARY OF THE INVENTION

The present invention provides a preparation method of an immunoaffinitychromatography column for Cryptococcus neoformans capsularpolysaccharide GXM. The affinity chromatography column is prepared byutilizing a monoclonal antibody of the Cryptococcus neoformans capsularpolysaccharide GXM.

Preferably, the preparation method comprises the following steps:

(1) dissolving a monoclonal antibody of Cryptococcus neoformans capsularpolysaccharide GXM in a solution, and mixing the monoclonal antibodywith an affinity chromatography substrate to form thin homogenate;

(2) washing the homogenate obtained in the step (1) with a cross-linkingbuffer solution, and centrifuging to obtain a mixture of the antibodyand the affinity chromatography substrate;

(3) resuspending the mixture of the antibody and the affinitychromatography substrate obtained in step (2) with the cross-linkingbuffer solution, adding a difunctional binding agent into the obtainedsuspension, incubating, uniformly mixing, and separating the liquid fromthe solid to obtain an affinity chromatography substrate-antibodyconjugated complex;

(4) washing the affinity chromatography substrate-antibody conjugatedcomplex obtained in the step (3) with a blocking solution to blockexcessive active groups in the chromatography substrate so as toterminate a cross-linking reaction;

(5) resuspending the affinity chromatography substrate-antibodyconjugated complex obtained in the step (4) in the blocking solution,incubating, and uniformly mixing; and

(6) packing: filling the affinity chromatography substrate-antibodyconjugated complex obtained in the step (5) after successfullycross-linked when detection into a chromatographic column, therebyobtaining the immunoaffinity chromatography column for the Cryptococcusneoformans capsular polysaccharide GXM.

Preferably, the method further comprises a step (7) of preservation:preserving the affinity chromatography substrate-antibody conjugatedcomplex obtained in the step (6) or the packed immunoaffinitychromatography column for the Cryptococcus neoformans capsularpolysaccharide GXM obtained in the step (6) under a condition of 4° C.,and enabling the performance to be stable within 1 year.

Preferably, the method further comprises a step (8) of regeneration:washing and regenerating the chromatography column preserved under thecondition of 4° C. in the step (7) or the used chromatography columnwith a buffer solution which has the same solvent as a to-be-purifiedsample, in an amount of 10-25 times of the volume of the column bedbefore use.

Preferably, the affinity chromatography substrate-antibody conjugatedcomplex obtained in the step (5) is subjected to preservative treatmentand filled into the chromatography column, wherein the preservativetreatment comprises the following steps: washing the affinitychromatography substrate-antibody conjugated complex obtained in thestep (5) with a PBS buffer solution, resuspending the complex in the PBSbuffer solution, and adding merthiolate for preservation. Theconcentration of the merthiolate is 0.005-0.015%.

Preferably, the affinity chromatography substrate in the step (1) isselected from protein A microbeads, protein G microbeads and activemicrobeads, and preferably protein A microbeads.

Preferably, the solution in the step (1) is selected from a carbonatebuffer solution with a pH value of 8.0-9.0.

Preferably, a ratio of the affinity chromatography substrate to thesolution in the step (1) is as follows: 0.5-2.0 mL of the affinitychromatography substrate is added into every 10 mL of the solution; aratio of the affinity chromatography substrate to the antibody is asfollows: every 1 mL of the affinity chromatography substrate is bound to1-4 mg of the monoclonal antibody; further preferably, a ratio of theaffinity chromatography substrate to the solution in the step (1) is asfollows: 1 mL of the affinity chromatography substrate is added intoevery 10 mL of the solution; and a ratio of the affinity chromatographysubstrate to the antibody is as follows: every 1 mL of the affinitychromatography substrate is bound to 2 mg of the monoclonal antibody.

Preferably, the cross-linking buffer solution in the step (2) is a0.1-0.3 mol/L of sodium borate solution with a pH value of 8.0-9.5, andthe use amount of the buffer solution is 5-15 times of the volume of theaffinity chromatography substrate; further preferably, the cross-linkingbuffer solution is a sodium borate solution with a concentration of 0.2mol/L and a pH value of 9.0, and the use amount of the buffer solutionis 10 times of the volume of the affinity chromatography substrate; andwashing frequency is 1-3 times, and a centrifugal condition is tocentrifuge for 2-5 minutes per 3000 g or to centrifuge for 30 secondsper 10000 g.

Preferably, the cross-linking buffer solution in the step (3) is 0.1-0.3mol/L of sodium borate solution with pH value of 8.3-9.5, and the useamount of the buffer solution is 5-15 times of the volume of theaffinity chromatography substrate; further preferably, the cross-linkingbuffer solution is a sodium borate solution with a concentration of 0.2mol/L and a pH value of 9.0, and the use amount of the buffer solutionis 10 times of the volume of the affinity chromatography substrate.

Preferably, the difunctional binding agent in the step (3) is selectedfrom dimethyl heptandilate, carbonyldimidazole, cyanogen bromide,hydroxysuccinimide and acetyl iodine, and preferably the dimethylheptandilate. The use amount of the difunctional binding agent is asfollows: the final concentration of the binding agent in affinitychromatography substrate suspension is 15-25 mmol/L, and preferably 20mmol/L.

Preferably, the blocking solution in the steps (4) and (5) is selectedfrom ethanol amine, aminoethane and other solutions of small molecularsubstances containing active groups which can be bound to amino, andpreferably an ethanol amine solution, further preferably an ethanolamine solution with a concentration of 0.1-0.25 mol/L and a pH value of7.5-8.5, and more preferably 0.2 mol/L of ethanol amine solution with apH value of 8.0.

Preferably, a detection process of cross-linking efficiency of theaffinity chromatography substrate-antibody conjugated complex in thestep (6) comprises the following steps: taking an affinitychromatography substrate sample and a sample obtained by cross-linkingthe affinity chromatography substrate and the antibody, respectivelyadding the samples into a LaemmLi buffer solution to boil, respectivelytaking out two samples equivalent to 1 mL and 9 mL, performingelectrophoresis in 10% of SDS-polyacrylamide gel, and staining withCoomassie brilliant blue, wherein if a heavy chain zone (55 kDa) ispresent in the sample before cross-linking while absent aftercross-linking, it indicates that cross-linking is successful.

Preferably, a container is rinsed with a PBS buffer solution afterpacking in the step (6) is completed, and the residual affinitychromatography substrate is collected. If possible, only the affinitychromatography substrate-antibody conjugated complex needed by the totalGXM in a to-be-purified sample is used.

The present invention further provides an immunoaffinity chromatographycolumn for Cryptococcus neoformans capsular polysaccharide GXM preparedby the above method.

The present invention further provides a preparation method of theCryptococcus neoformans capsular polysaccharide GXM. The preparationmethod comprises a step of purifying GXM by utilizing the above GXMimmunoaffinity chromatography column.

Preferably, the preparation method comprises the following steps:

(1) performing crude extraction on Cryptococcus neoformans capsularpolysaccharide GXM; and

(2) purifying a crude extract of the Cryptococcus neoformans capsularpolysaccharide GXM by utilizing the GXM immunoaffinity chromatographycolumn for the Cryptococcus neoformans capsular polysaccharide.

The crude extraction of the Cryptococcus neoformans capsularpolysaccharide GXM comprises the following steps:

(1) culturing Cryptococcus neoformans until a bacteria concentrationreaches a later logarithmic phase;

(2) performing autoclaved sterilization on bacterium liquid,centrifuging to remove thalli, and reserving supernatant;

(3) slowly adding calcium acetate powder into the supernatant whilestirring, and adding glacial acetic acid to regulate the pH value to beacidic; and

(4) adding ethanol into the solution obtained in the step (3), standingovernight at 4° C., centrifuging, removing the supernatant, drying andprecipitating, thereby obtaining the crude extract of the Cryptococcusneoformans capsular polysaccharide GXM.

Preferably, culture conditions of Cryptococcus neoformans in the step(1) of performing crude extraction on the Cryptococcus neoformanscapsular polysaccharide GXM are as follows: a culture medium is a YMbouillon culture-medium, a culture temperature is 30° C., a shake speedis 200 rpm, culture time is 32-40 hours, and a formula of the YMbouillon culture-medium is that every liter of the culture mediumcontains 3 g of yeast extract, 5 g of peptone, 3 g of maltose extractand 10 g of D-glucose.

Preferably, the addition amount of the calcium acetate powder in thestep (3) is as follows: the final concentration of the calcium acetatepowder in the supernatant reaches 2-8%, and the pH value is regulated to4.6-5.2 by the glacial acetic acid.

Preferably, the concentration of the ethanol in the step (4) is 90-98%,and the addition amount is 2-4 times of the volume of the solutionobtained in the step (3).

The step of purifying the crude extract of the Cryptococcus neoformanscapsular polysaccharide GXM by utilizing the immunoaffinitychromatography column for the Cryptococcus neoformans capsularpolysaccharide GXM comprises the following steps:

(1) rinsing the immunoaffinity chromatography column for theCryptococcus neoformans capsular polysaccharide GXM by utilizing abuffer solution which is the same as a to-be-purified sample solution;

(2) enabling the to-be-purified sample solution to flow through thechromatography column treated in the step (1);

(3) washing the column with a binding buffer solution;

(4) washing the column with a pre-elution buffer solution;

(5) performing stepwise elution, continuously enabling an elution buffersolution to flow through the chromatography column, and collecting eachcomponent in different tubes; and

(6) detecting the content of the GXM in each tube, and merging all thetubes with high concentrations.

Preferably, (7) enabling an initial buffer solution in an amount of10-25 times of a column bed volume to flow through the chromatographycolumn to regenerate, adding merthiolate to enable the finalconcentration of the buffer solution to be 0.005-0.015%, and preservingthe chromatography column under the condition of 4° C.

Preferably, flow velocity of the to-be-purified sample in the step (2)is 0.5-1.5 mL/h. The flow velocity is controlled by a pump, andpreferably a peristaltic pump.

Preferably, the binding buffer solution in the step (3) is selected fromone of a PBS buffer solution with a pH value of 6.0-8.0, a Tris-HClbuffer solution with a pH value of 6.0-8.0 and an acetic acid-sodiumacetate buffer solution with a pH value of 6.0-8.0, and preferably thePBS buffer solution with the pH value of 6.0-8.0. The use amount of thebinding buffer solution in the step (3) is 10-25 times of the column bedvolume, and preferably 20 times of the column bed volume.

Preferably, the pre-elution buffer solution in the step (4) is acarbonate buffer solution with a pH value of 8.5-9.5. The use amount ofthe pre-elution buffer solution is 10-25 times of the column bed volume,and preferably 20 times of the column bed volume.

Preferably, the elution buffer solution in the step (5) is a buffersolution with a pH of 3.0, preferably 0.1M glycine buffer solution witha pH of 3.0, a citrate-phosphate buffer solution with a pH of 3.0, acitrate-sodium citrate buffer solution with a pH of 3.0 and anacetate-sodium acetate buffer solution with a pH of 3.0. The use amountof the elution buffer solution is 0.4-0.8 of the column bed volume.

Preferably, the GXM eluant obtained in the step (6) is subjected todialysis desalting or the pH value of the GXM eluant is regulated by abuffer solution.

The present invention provides a Cryptococcus neoformans capsularpolysaccharide GXM prepared by the above method.

The present invention further provides applications of theimmunoaffinity chromatography column for the Cryptococcus neoformanscapsular polysaccharide GXM in purification of the Cryptococcusneoformans capsular polysaccharide GXM and early diagnosis ofcryptococcus neoformans meningitis.

The present invention further provides an application of theCryptococcus neoformans capsular polysaccharide GXM in an earlydiagnosis reagent for cryptococcus neoformans meningitis.

The present invention further provides a polyclonal antibody whichresists Cryptococcus neoformans capsular polysaccharide GXM. Thepolyclonal antibody is prepared by the following method: immunizinganimal-derived serum by taking GXM as an immunogen, and separating andpurifying to obtain an antibody. A purification method is a saturatedammonium sulfate precipitation method and/or an affinity chromatographymethod. The immunization method may be subcutaneous injection,intrasplenic injection, intravenous injection or intraperitonealinjection. An immunizing dosage is 0.01-1 mg per animal. Immunizedanimals are rats, mice, guinea pigs, rabbits, chickens, sheep, horses,pigs or donkeys, and preferably rabbits.

A preparation method of the GXM polyclonal antibody comprises thefollowing specific steps:

(1) immunizing an animal by using a GXM antigen;

(2) determining a serum titer of the immunized animal, and collectingblood from the immunized animal; and

(3) performing preliminary purification by using a saturated ammoniumsulfate precipitation method and/or an affinity chromatography method.

The anti-GXM polyclonal antibody prepared by the above steps may bespecifically bound to GXM and is displayed as a homogenous antibodyproduct by using SDS-PAGE. Indirect ELISA detection shows that a titerof the polyclonal antibody is greater than 1:1×10⁶.

The present invention further provides a GXM antigen immunoassay kit forCryptococcus neoformans capsular polysaccharide. The kit comprises a GXMcoated enzyme-labeled carrier, an anti-GXM polyclonal enzyme-labeledantibody and a GXM standard substance.

The GXM standard substance is composed of a series of GXM solutions withdifferent concentrations. The GXM solutions are prepared by dissolvingGXM into an appropriate solvent to form GXM solutions with specificconcentrations.

The GXM coated enzyme-labeled carrier is prepared by the followingmethod:

{circle around (1)} diluting GXM with a coating buffer solution toobtain a GXM coating solution, and adding the GXM coating solution intowells of an ELISA plate for coating;

{circle around (2)} adding a blocking solution into the wells of theELISA plate obtained in the step {circle around (1)} for blocking; and

{circle around (3)} removing the blocking solution from the ELISA plateobtained in the step {circle around (2)}, and incubating to obtain theGXM coated enzyme-labeled carrier.

Preferably, the enzyme-labeled carrier is a microwell plate or a plastictube, and preferably the enzyme-labeled carrier is the microwell plate.

Preferably, an enzyme used for labeling is horse radish peroxidase (HRP)or alkaline phosphatase (AP), and preferably, the enzyme used forlabeling is the HRP.

Preferably, the GXM standard substance at least has 3 differentconcentrations in a range of 0-100 ng/mL.

In a specific embodiment of the present invention, in the above antigenimmunoassay kit for the Cryptococcus neoformans capsular polysaccharide,the GXM standard substance has 5 different concentrations marked as a,b, c, d and e respectively. The concentrations are respectively 100, 32,10, 6.4 and 3.2 ng/mL.

Preferably, the antigen immunoassay kit for the Cryptococcus neoformanscapsular polysaccharide further comprises a sample treatment solution, awash concentrate, a sample diluent, a substrate solution and a stopsolution.

Preferably, components and ratios of the wash concentrate, the samplediluent, the sample treatment solution, the substrate solution and thestop solution are as follows:

the wash concentrate: a phosphate buffer solution containing 0.4-1.0% ofTween;

the sample diluent: artificial cerebrospinal fluid or artificial serum;

the sample treatment solution is selected from the following solutions:a protein denaturation solution with a pH of 2.0-10.0, 0.05-0.2 mol/L ofEDTA (ethylene diamine tetraacetic acid disodium salt) solution with apH of 4.0-4.8, 0.07-0.2 mol/L of glycine-HCL (glycine-hydrochloric acid)solution with a pH of 2.2-2.8, 0.05-15 mg/mL of pronase with a pH of8.0-9.0, and 0.01-0.1 mol/L of SDS (sodium dodecyl sulfate) solutionwith a pH of 8.5-10.0 or 1-8 mol/L of urea with a pH of 7.2-8.0;

the substrate solution comprises TMB (Tetramethylbenzidine), OPD(o-Phenylenediamine), OT (o-Tolidine) and ABTS(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) or p-NNP(p-Nitrophenyl Phosphate), and preferably the TMB; and

the stop solution is 1-20 mol/L of sulfuric acid solution, andpreferably 2 mol/L of sulfuric acid solution.

In a specific embodiment of the present invention, the wash concentratecomprises the following components in parts by weight: 96.0 parts ofsodium chloride, 2.40 parts of potassium chloride, 42.96 parts of sodiumphosphate dibasic dodecahydrate, 2.88 parts of monopotassium phosphate,0.05 part of Tween-20 and 1000 parts of ultrapure water.

The present invention further provides a preparation method of the GXMantigen immunoassay kit for the Cryptococcus neoformans capsularpolysaccharide GXM. The preparation method comprises the followingspecific steps:

(1) Preparing an Enzyme-Labeled Carrier

{circle around (1)} diluting GXM with a coating buffer solution toobtain a GXM coating solution, and adding the GXM coating solution intowells of an ELISA plate for coating;

{circle around (2)} adding a blocking solution into the wells of theELISA plate obtained in the step {circle around (1)} for blocking; and

{circle around (3)} removing the blocking solution from the ELISA plateobtained in the step {circle around (2)}, and incubating to obtain a GXMcoated enzyme-labeled carrier;

(2) Preparing a GXM Standard Substance

diluting a GXM antigen, and preparing the GXM standard substance; and

(3) Preparing an Anti-GXM Polyclonal Enzyme-Labeled Antibody Solution

An enzyme used for labeling is the horse radish peroxidase, and ananti-GXM polyclonal enzyme-labeled antibody is prepared by a periodateoxidation method; or the enzyme used for labeling is the alkalinephosphatase, and the anti-GXM polyclonal enzyme-labeled antibody isprepared by a glutaraldehyde cross-linking method.

The anti-GXM polyclonal enzyme-labeled antibody is diluted by an enzymeconjugate stabilizer to prepare the anti-GXM polyclonal enzyme-labeledantibody solution. Preferably, the preparation method of the antigenimmunoassay kit for the Cryptococcus neoformans capsular polysaccharidecomprises the following specific steps:

(1) preparing an enzyme-labeled carrier

{circle around (1)} diluting GXM to 25-2000 ng/100 μL with a coatingbuffer solution to obtain a GXM coating solution, adding the GXM coatingsolution into wells of an ELISA plate, respectively adding 60-200 μL ofthe coating solution into each well, and coating the ELISA plate at 2-8°C. for 8-16 h;

{circle around (2)} adding a blocking solution into the wells of theELISA plate obtained in the step {circle around (1)}, respectivelyadding 60-200 μL of the blocking solution into each well, and blockingat 37° C. for 30-90 min; and

{circle around (3)} removing the blocking solution from the ELISA plateobtained in the step {circle around (2)}, and standing at a constanttemperature of 37° C. for 30-90 min, thereby obtaining a GXM coatedenzyme-labeled carrier;

(2) preparing a GXM standard substance

diluting a GXM antigen into at least 3 different concentrations in arange of 0-100 ng/mL;

(3) preparing an anti-GXM polyclonal enzyme-labeled antibody solution:

an enzyme used for labeling is the horse radish peroxidase, and ananti-GXM polyclonal enzyme-labeled antibody is prepared by a periodateoxidation method; or the enzyme used for labeling is the alkalinephosphatase, and the anti-GXM polyclonal enzyme-labeled antibody isprepared by a glutaraldehyde cross-linking method.

The anti-GXM polyclonal enzyme-labeled antibody is diluted by an enzymeconjugate stabilizer according to a ratio of 1:2000-1:20000 to preparethe anti-GXM polyclonal enzyme-labeled antibody solution;

(4) preparing a sample treatment solution, a wash concentrate, a samplediluent, a substrate solution and a stop solution;

More preferably, the preparation method of the antigen immunoassay kitfor the Cryptococcus neoformans capsular polysaccharide furthercomprises:

(5) packaging the anti-GXM polyclonal enzyme-labeled antibody, the GXMstandard substance, the sample treatment solution, the wash concentrate,the sample diluent, the substrate solution and the stop solution, andplacing the above solutions into a kit box together with a GXM-coatedenzyme-labeled antibody and a sealing film.

Preferably, the coating buffer solution is selected from 0.01-0.20 mol/Lof PBS (phosphate) buffer solution with a pH of 7.0-8.0, 0.05-0.20 mol/Lof CBS (carbonate) buffer solution with a pH of 9.0-9.6 or 0.05 mol/L ofTris(hydroxy methyl aminomethane) buffer solution with a pH of10.0-10.6.

Preferably, the blocking solution comprises the following component:0.01-0.20 mol/L of PBS buffer solution with a pH of 7.0-8.0 containing3-5% of skim milk powder or 1-4% of BSA (Bovine Serum Albumin).

In a specific embodiment of the present invention, the GXM standardsubstance is prepared by diluting GXM with the sample diluent, and has 5different concentrations marked as a, b, c, d and e respectively. Theconcentrations are respectively 100, 32, 10, 6.4 and 3.2 ng/mL.

Another purpose of the present invention is to provide an application ofthe antigen immunoassay kit for the Cryptococcus neoformans capsularpolysaccharide in detection of GXM concentrations.

Preferably, the application of the antigen immunoassay kit for theCryptococcus neoformans capsular polysaccharide in detection of GXMconcentrations is a competitive ELISA method (one-step method),comprising the following specific steps:

(1) mixing a to-be-detected sample and a sample treatment solutionaccording to a volume ratio of 1:1-5:1, boiling for 1-10 min, andcentrifuging to obtain a to-be-detected substance;

(2) uniformly mixing the to-be-detected substance in the step (1) and ananti-GXM polyclonal enzyme-labeled antibody according to an equivalentvolume, adding the mixture into a GXM-coated ELISA plate, incubating for20-60 min, and washing the plate after incubating; and

(3) adding 50-100 μL of substrate solution into the ELISA plate in thestep (2) for developing, adding the stop solution, and detectingabsorbance.

Preferably, the application of the antigen immunoassay kit for theCryptococcus neoformans capsular polysaccharide in detection of GXMconcentrations is a competitive ELISA method (two-step method),comprising the following specific steps:

(1) mixing a to-be-detected sample and a sample treatment solutionaccording to a volume ratio of 1:1-5:1, boiling for 1-10 min, andcentrifuging to obtain a to-be-detected substance;

(2) uniformly mixing the to-be-detected matter in the step (1) and ananti-GXM polyclonal enzyme-labeled antibody according to an equivalentvolume, and incubating for 20-60 min;

(3) adding the mixture in the step (2) into a GXM-coated ELISA plate,incubating for 20-60 min, and washing the plate after incubating; and

(4) adding 50-100 μL of substrate solution into the ELISA plate in thestep (3) for developing, adding the stop solution, and detectingabsorbance.

An appliance principle of the antigen immunoassay kit for theCryptococcus neoformans capsular polysaccharide is as follows:

1) coating GXM onto a solid phase carrier to prepare a solid phaseantigen;

2) mixing a treated to-be-detected sample and an anti-GXM polyclonalenzyme-labeled antibody, and reacting at a constant temperature;

3) adding a mixed solution in the step 2) into the solid phase in the1), and enabling a to-be-detected antigen and a coating antigen to becompetitively bound to limited antibody binding sites;

4) carrying out a constant temperature reaction, completely washing, andadding an enzymatic reaction substrate TMB for developing, wherein thecolor is in negative correlation to GXM concentrations in theto-be-detected sample; and

5) determining absorbance (value A) under a certain wavelength by usinga microplate reader, thereby realizing detection of the antigen througha standard curve.

The Cryptococcus neoformans adopted in the present invention ispurchased from China Pharmaceutical Culture Collection Center.

The capsular polysaccharide monoclonal antibody adopted in the presentinvention is provided by Dyna (Tianjin) Biological Technology Co., Ltd.

According to the preparation method of the Cryptococcus neoformanscapsular polysaccharide GXM provided by the present invention, the crudeextract of the Cryptococcus neoformans capsular polysaccharide GXM ispurified by using immunoaffinity chromatography, so that the use of atoxic chemical reagent is effectively avoided, the safety of an operatoris ensured, environmental pollution is avoided, specificity is high, anda high-purity Cryptococcus neoformans capsular polysaccharide may beprepared while simplifying the preparation process. The prepared GXM isused as an immunogen for producing the polyclonal antibody, and theprepared polyclonal antibody has the characteristics of high titer andexcellent specificity, is stable in property and has excellentapplication prospects. The GXM antigen immunoassay kit is prepared byutilizing the GXM and the anti-GXM polyclonal antibody. The kit adopts acompetition method, and the competition method comprises the followingsteps: coating the GXM onto the ELISA plate at first, enabling theto-be-detected sample or a standard antigen and the coating antigen tobe competitively bound with limited antibody binding sites, carrying outa color development reaction with a substrate by virtue of the enzyme,and detecting and calculating to obtain an antigen concentration. Thedetection kit has good sensitivity, specificity, repeatability andstability, has high recovery rate of a target compound and may providemore accurate and reliable inspection results. The kit is simple andfeasible in use and operation, rapid and sensitive in detection and lowin price and provides an effective tool for clinical detection of theGXM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a quantitative detection result of a Cryptococcus neoformanscapsular polysaccharide GXM provided in the present invention;

FIG. 2 is an HPLC purity analysis result of a Cryptococcus neoformanscapsular polysaccharide GXM provided in the present invention;

FIG. 3 shows an SDS-PAGE impure protein content analysis result of aCryptococcus neoformans capsular polysaccharide GXM provided in thepresent invention;

FIG. 4 shows an SDS-PAGE detection result of a rabbit IgG typepolyclonal antibody provided in the present invention;

FIG. 5 shows a titer determination result of a rabbit IgG typepolyclonal antibody provided in the present invention; and

FIG. 6 shows a standard curve graph of a GXM immunoassay kit provided inthe present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In order to make purposes, technical means and advantages of the presentinvention clearly understood, the present invention is further describedin detail below in combination with drawings.

The above only describes preferred embodiments of the present invention,not intended to limit a protection scope of the present invention. Allmodifications, equivalent replacements, improvements and the like madewithin spirit and principles of the present invention should be includedin the protection scope of the present invention.

Embodiment 1

Extraction of a Cryptococcus neoformans capsular polysaccharide GXM,comprising the following specific steps:

(1) preparing 0.6 L of YM bouillon culture-medium containing thecomponents of 1.8 g of yeast extract, 3 g of peptone, 1.8 g of maltoseextract and 6 g of D-glucose, and culturing Cryptococcus neoformans,wherein a culture temperature is 30° C., a shake speed is 200 rpm, andculture time is 36 h;

(2) sterilizing bacterium liquid at 121° C. for 25 min, and centrifugingfor 30 min per 16000 g to remove thalli;

(3) slowly adding calcium acetate powder into the supernatant whilestirring until a final concentration is 5%, and adding glacial aceticacid to regulate a pH value to be about 5.0; and

(4) adding 95% of ethanol in an amount of three times of a volume of thesolution into the solution, immediately producing a precipitate, andstanding overnight at 4° C.; dumping most of the supernatant,transferring residual substances into 50 mL of round bottom centrifugetube, centrifuging for 5 min per 12000 g, removing the supernatant,precipitating and drying, thereby obtaining the crude extract of theCryptococcus neoformans capsular polysaccharide GXM.

Embodiment 2

Preparation of an immunoaffinity chromatography column for aCryptococcus neoformans capsular polysaccharide GXM, comprising thefollowing specific steps:

(1) binding a monoclonal antibody of Cryptococcus neoformans capsularpolysaccharide GXM onto protein A microbeads, mixing the antibody andthe protein A into thin homogenate according to the rate that about 2 mgof the monoclonal antibody may be bound to every milliliter of wetmicrobeads, adding about 1 mL of microbeads into a solution with a totalvolume of 10 mL, incubating at a room temperature for 1 h, slightlyshaking and mixing to be uniform;

(2) washing the microbeads with 0.2 mol/L of sodium borate (pH of 9.0)in an amount of 10 times of the volume for 2 times, and centrifuging for2 min per 3000 g each time, or centrifuging for 30 s per 10000 g;

(3) resuspending the microbeads with the 0.2 mol/L of sodium borate (pHof 9.0) in an amount of 10 times of the volume, reserving a sampleequivalent to 10 mL of the wet microbeads, adding an enough amount ofdimethyl heptandilate (solid) into the microbead homogenate to enable afinal concentration to be 20 mmol/L, incubating at the room temperaturefor 30 min, enabling the antibody bound onto the protein A microbeads tobe cross-linked with a microbead substrate, slightly mixing to beuniform to obtain a microbead-antibody cross-linked complex, andreserving a sample equivalent to 10 mL of the cross-linked complex;

(4) washing the microbead-antibody cross-linked complex with 0.2 mol/Lof ethanol amine (pH of 8.0) once so as to terminate the cross-linkingreaction;

(5) resuspending the microbead-antibody cross-linked complex obtained inthe step (4) in the 0.2 mol/L of ethanol amine solution, incubating atthe room temperature for 2 h, and slightly mixing to be uniform;

(6) washing the microbead-antibody cross-linked complex obtained in thestep (5) with PBS, resuspending in the PBS, and adding merthiolate untilthe final concentration is 0.01% for preserving; and

(7) after the microbead-antibody cross-linked complex is successfullycross-linked when detected, filling the microbead-antibody cross-linkedcomplex obtained in the step (6) into a chromatographic column to obtainthe immunoaffinity chromatography column for the Cryptococcus neoformanscapsular polysaccharide, rinsing a container with the PBS, andcollecting residual microbeads. If possible, only the antibody microbeadsubstrate needed by the total GXM in a to-be-purified product is used.

Embodiment 3

Purification of the crude extract of the Cryptococcus neoformanscapsular polysaccharide GXM prepared in Embodiment 1, comprising thefollowing specific steps:

(1) taking the immunoaffinity chromatography column for the Cryptococcusneoformans capsular polysaccharide GXM prepared in Embodiment 2, andwashing the column with an initial buffer solution in an amount of 20times of a column bed volume;

(2) adding a to-be-purified sample solution into the chromatographycolumn, enabling the sample solution to flow through the chromatographycolumn according to flow velocity of being about 1 mL/h per milliliterof the column volume, and controlling the flow velocity by a peristalticpump;

(3) washing the column with a binding buffer solution in an amount of 20times of the column bed volume;

(4) washing the column with a pre-elution buffer solution in an amountof 20 times of the column bed volume;

(5) adopting stepwise elution, continuously enabling an elution buffersolution in an amount of 0.5 time of the column bed volume to passthrough the chromatography column, and collecting each component indifferent tubes;

(6) detecting content of the GXM in each tube, merging all the tubeswith high concentrations, and dialyzing collected antigen eluantaccording to the purpose of the antigen; and

(7) enabling an initial buffer solution in an amount of 20 times of acolumn bed volume to flow through a substrate, enabling thechromatography column to regenerate, adding 0.01% of merthiolate intothe buffer solution, and preserving the chromatography column in anenvironment of 4° C.

The initial buffer solution, the binding buffer solution, thepre-elution buffer solution and the elution buffer solution arerespectively high purity water, a PBS buffer solution with a pH of 7, acarbonate buffer solution with a pH of 9 and 0.1M of glycine buffersolution with a pH of 3.0.

Embodiment 4

Detection of the Cryptococcus neoformans capsular polysaccharide GXMprepared in Embodiment 3:

A. Determination of sugar content by a Dubois-sulfuric acid-phenolmethod, comprising the following specific steps:

(1) weighing 1 g of glucose and dissolving the glucose into 100 mL ofdH₂O, and preparing 1% of glucose solution;

(2) taking seven 100 mL of clean test tubes, and respectively adding 1mL of dH₂O and 25 μL of phenol solution into each test tube;

(3) respectively adding 0, 2.5, 5, 10 and 15 μL of glucose solutions and10 μL of to-be-detected solution into all the test tubes;

(4) slowly adding 2.5 mL of concentrated sulfuric acid into each tube,and uniformly mixing in a unified manner after all the test tubes arefilled with the concentrated sulfuric acid;

(5) placing the test tubes in a water bath for about 20 minutes, andcooling to a room temperature;

(6) carefully pouring liquid in each tube into a glass cuvette;

(7) determining an absorbance value under 485 nm by using an ultravioletspectrophotometer;

(8) drawing a standard curve; and

(9) calculating a polysaccharide concentration of the to-be-detectedsolution according to the standard curve.

The standard curve is shown in FIG. 1, has excellent linearity, and canbe used to calculate the concentration of the to-be-detected sample.

B. HPLC purity analysis, comprising the following specific steps:

taking 1 mL of a Cryptococcus neoformans capsular polysaccharide GXMsample (3.65 mg/mL)

chromatographic column: Sugar—ParkT72011A09P/N85188Waters

mobile phase: ultrapure water

flow velocity: 0.5 mL/min

column temperature: 85° C.

detector: a differential refraction detector

sensitivity setting range: 1.0×10⁻⁶-5.0×10⁻⁴ RIU

differential range: 1.00-1.75RIU

linear dynamic range: 5×10⁻³-5×10⁻⁹RIU

noise: <2×10⁻⁸RIUATTN

HPLC sample injection is detected by the differential refractiondetector. A result is shown in FIG. 2. If a single absorption peakappears, it indicates that purity of the sample is good.

C. SDS-PAGE impure protein content analysis, comprising the followingspecific steps:

(1) preparing SDS-PAGE gel with a concentration of 8%, and makingcolloid;

(2) adding 10 μL of the Cryptococcus neoformans capsular polysaccharideGXM sample prepared in Embodiment 3 into each well;

(3) performing electrophoresis under a constant voltage of 60V for 10min, then performing electrophoresis under a constant voltage of 120Vfor about 1 hour, and stopping electrophoresis until a bromophenol bluestripe is 1 cm away from a lower edge of a colloid plate;

(4) staining with Coomassie brilliant blue for 1 hour;

(5) decolorizing overnight at 4° C.; and

(6) shooting and observing.

A colloid figure is shown in FIG. 3. By contrast, the Cryptococcusneoformans capsular polysaccharide sample prepared in Embodiment 3 hashigh purity and does not contain any impure protein.

Embodiment 5

Verification of repeatability, recovery rate and stability of animmunoaffinity chromatography column for Cryptococcus neoformanscapsular polysaccharide GXM:

A. repeatability verification, comprising the following specific steps:

(1) preparing 5 immunoaffinity chromatography columns for theCryptococcus neoformans capsular polysaccharide GXM according to thesteps in Embodiment 2, and respectively labeling the chromatographycolumns as 001, 002, 003, 004 and 005;

(2) taking a crude Cryptococcus neoformans capsular polysaccharide GXMprepared in Embodiment 1 as a to-be-purified sample, and uniformlydividing the sample into five parts;

(3) respectively adding the 5 parts of 2 mL of the to-be-purified sampleinto the 5 chromatography columms in parallel test, and purifyingaccording to the operation in Embodiment 3; and

(4) identifying the purified polysaccharide GXM according to theoperation in Embodiment 4. Results are as follows:

Quantitative results of the sulfuric acid-phenol method are shown inFIG. 1. Total CV of the purified polysaccharide is less than 10%.

The single absorption peak appears in the HPLC purity analysis.

Any impure protein band does not appear during the SDS-PAGE impureprotein content analysis.

TABLE 1 Repeatability verification results of the immunoaffinitychromatography column for the crytococcus neoformans capsularpolysaccharide GXM Chromatography column number 001 002 003 004 005Total sugar 19.65 18.05 21.89 19.12 18.56 content Mean 19.56 value SD1.48 CV 7.65%

B. recovery verification, comprising the following specific steps:

(1) preparing 5 immunoaffinity chromatography columns for theCryptococcus neoformans capsular polysaccharide GXM according to thesteps in Embodiment 2;

(2) taking a Cryptococcus neoformans capsular polysaccharide GXMprepared in Embodiment 3 as a purification standard sample with aconcentration of 3.65 mg/mL;

(3) respectively adding 2 mL of purification standard sample solutioninto the 5 chromatography columns in parallel test, and purifyingaccording to the operation in Embodiment 3; and

(4) detecting total sugar content of the purified standard sample,wherein results are shown in Table 2, and the recovery rate iscalculated to be between 96.5% and 100.5%.

TABLE 2 Recovery rate verification results of the immunoaffinitychromatography column for the crytococcus neoformans capsularpolysaccharide GXM Chromatography column number 001 002 003 004 005 Meanvalue Total 7.18 7.34 7.09 7.05 7.13 7.16 sugar content Recovery 98.36%100.5% 97.12% 96.57% 97.67% 98.08% rate

C. stability verification, comprising the following specific steps:

(1) preparing 1 immunoaffinity chromatography column for theCryptococcus neoformans capsular polysaccharide GXM according to thesteps in Embodiment 2;

(2) taking a Cryptococcus neoformans capsular polysaccharide GXMprepared in Embodiment 3 as a purification standard sample with aconcentration of 3.65 mg/mL;

(3) adding 1 mL of purification standard sample solution into thechromatography column, purifying according to the operation inEmbodiment 3, and performing repetitive sample injection on the samecolumn for 10 times, (regenerating after elution each time); and

(4) detecting total sugar content of the purified standard sample after10 times, wherein results are shown in Table 3, and the recovery rate iscalculated to be between 78.08% and 105.80%.

TABLE 3 Stability verification results of the immunoaffinitychromatography column for the crytococcus neoformans capsularpolysaccharide GXM Purification Total sugar Recovery frequency contentrate 1 3.47 95.10% 2 3.64 99.70% 3 3.86 105.80% 4 3.32 91.00% 5 3.5797.80% 6 3.12 85.50% 7 3.45 94.50% 8 3.01 82.50% 9 3.05 83.60% 10 2.8578.08% Mean value 3.334 SD 0.32 CV 9.6%

It is known from the above results that, the immunoaffinitychromatography column for the Cryptococcus neoformans capsularpolysaccharide GXM prepared by the steps in Embodiment 2 has extremelyhigh specificity and recovery rate on the target compound, is excellentin the repeatability and the stability, and can achieve the effects ofeffectively decreasing purification and preparation cost, simplifyingpreparation steps and increasing preparation efficiency when used inpurification and preparation of the Cryptococcus neoformans capsularpolysaccharide GXM.

Embodiment 6 Biological Activity Contrast of Cryptococcus neoformansCapsular Polysaccharide GXM

Sample: the Cryptococcus neoformans capsular polysaccharide GXM preparedin Embodiment 3

Reference substance: Cryptococcus neoformans capsular polysaccharideprepared by a method disclosed in a patent literature CN201110240065.X.

Contrast and verification of the biological activity of the Cryptococcusneoformans capsular polysaccharide GXM are performed, comprising thefollowing specific steps:

A. titer verification, comprising the following specific steps:

(1) coating capsular polysaccharide purified in two manners onto anELISA plate according to 10 ng/well, 25 ng/well, 50 ng/well and 100g/well;

(2) diluting the HRP-labeled antibody of the monoclonal antibody for theCryptococcus neoformans capsular polysaccharide GXM according to theratios of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and1:128000, adding the antibody onto the ELISA plate coated by thecapsular polysaccharide, and incubating at 37° C. for 60 min;

(3) washing: removing liquid in the ELISA plate wells, adding not lessthan 300 μL of working washing solution into each well each time,standing for 40 s, patting to be dry, repeating the above washingoperation, and washing for 5 times;

(4) developing: adding 50 μL of substrate solution TMB into each wellafter washing, incubating at 37° C. for 15 min, and keeping out of thesun;

Stopping: adding 50 μL of stop solution into each well, mixing to beuniform, and reading an absorbance value at 450 nm.

TABLE 4 Titer verification results of the crytococcus neoformanscapsular polysaccharide GXM Coating quantity Dilution Sample Referencesubstance ratio 10 ng/well 25 ng/well 50 ng/well 100 ng/well 10 ng/well25 ng/well 50 ng/well 100 ng/well blank 0.050 0.048 0.047 0.051 0.0520.045 0.046 0.049 1:2000 3.630 4.000 4.000 4.000 2.163 2.562 2.782 2.9411:4000 2.976 3.976 4.000 4.000 1.082 1.281 1.391 1.471 1:8000 1.8153.750 4.000 4.000 0.541 0.641 0.696 0.735 1:16000 0.908 2.287 3.2183.462 0.270 0.320 0.348 0.368 1:32000 0.454 1.143 2.516 2.767 0.1350.160 0.174 0.184 1:64000 0.227 0.572 1.258 1.384 0.068 0.080 0.0870.092 1:128000 0.113 0.286 0.629 0.692 0.034 0.040 0.043 0.040

It is known from the above results that, the titer of the sample isobviously higher than that of the latter. It is indicated that, bindingcapacity between the GXM antigen prepared by the method in the presentapplication and the antibody is far higher than that in the prior art.

B. Verification of biological activity, comprising the followingspecific steps:

(1) preparing a GXM immunoassay kit;

(2) respectively diluting the sample and the reference substance to be1000 ng/mL, 500 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 20 ng/mL and 10ng/mL; and

(3) detecting by using the GXM immunoassay kit.

TABLE 5 Biological activity verification results of the crytococcusneoformans capsular polysaccharide GXM Sample Reference substanceConcentration Recovery Recovery Recovery Recovery ng/mL concentrationrate concentration rate 1000 1078 107.8% 215.6 21.6% 500 537 107.4%107.4 21.5% 200 228 114.0% 45.6 22.8% 100 107 107.0% 21.4 21.4% 50 52104.0% 10.4 20.8% 20 25 125.0% 5 25.0% 10 12 120.0% 2.4 24.0%

It is known from Table 5 that, the recovery rate of the sample is farhigher than that of the reference substance.

Embodiment 7: Preparation of Anti-GXM Polyclonal Antibody

1. Immunizing Animals

steps: performing equivalent volume mixing on a GXM antigen and aFreund's complete adjuvant to an appropriate volume, fully emulsifying,performing subcutaneous multi-point injection on New Zealand big earrabbits, controlling an immunizing dosage of each rabbit to be 0.01-1mg, collecting ear blood 3 days before immunization, separating serum toperform negative control, and immunizing once every 2 weeks afterprimary immunization, wherein the method is the same as that in thefirst time.

2. Acquisition of a Polyclonal Antibody

1) titer determination: collecting blood to determine the titer onceevery a few days during and after immunization, wherein immunizationfrequency is not less than 3 times; and

2) separating antiserum: collecting lots of blood by using a carotidartery bloodletting method when a serum titer reaches the highest value,centrifuging at a high speed after blood coagulation and serumseparation, taking supernatant, and preserving at −20° C.

3. Performing Preliminary Purification by Using a Saturated AmmoniumSulfate Salting-Out Method

(1) taking 2 mL of antiserum sample, adding physiological saline of anequivalent volume, then adding 4 mL of saturated ammonium sulfatesolution, and precipitating overnight at 4° C.;

(2) centrifuging at low temperature for 10 min per 10000 g, removing thesupernatant, dissolving the precipitate with 2 mL of PBS, slowlydripping 1 mL of the saturated ammonium sulfate solution, and standingat 4° C. for 1 hour, and

(3) centrifuging at low temperature for 10 min per 10000 g, removing thesupernatant, dissolving the precipitate with 1 mL of PBS, and dialyzingovernight at 4° C. with a PBS solution.

4. Further Purifying by Affinity Chromatography Method

(1) washing the column with an elution buffer solution (0.01 mol/L ofphosphatic buffer solution with a pH of 7.4) in an amount of 5-10 timesof a column bed volume;

(2) washing the column with a coupling buffer solution (0.01 mol/L ofphosphate buffer solution with a pH of 7.4) in an amount of 5-10 timesof the column bed volume;

(3) loading the sample subjected to preliminary purification by usingthe saturated ammonium sulfate salting-out method;

(4) washing the column with the coupling buffer solution (0.01 mol/L ofphosphate buffer solution with the pH of 7.4) in an amount of 5-10 timesof the column bed volume; and

(5) eluting with an elution buffer solution (a glycine-hydrochloric acidbuffer solution with a pH of 2.8) in an amount of 2-5 times of thecolumn bed volume, thereby obtaining the polyclonal antibody resistingthe Cryptococcus neoformans capsular polysaccharide GXM antigen.

Embodiment 8: Detection of an Anti-GXM Polyclonal Antibody

1. SDS-PAGE Electrophoresis Detection

The anti-GXM polyclonal antibody prepared in Embodiment 7 is subjectedto SDS-PAGE electrophoresis, and obtained gel is stained with Coomassiebrilliant blue. Experimental results are shown in FIG. 4 (a pAb lane isan anti-GXM polyclonal antibody prepared in Embodiment 6, and an M laneis a protein Marker). It can be seen from the figure that, clear bandsrespectively appear in molecular weight areas of 25 kD and 50 kD and arerespectively a hydrocarbon chain and a heavy chain of an antibodyprotein. Any impure protein band does not appear. It indicates that, theanti-GXM polyclonal antibody prepared in Embodiment 7 has extremely highpurity.

2. Titer Determination

The antibody titer is determined by using an indirect ELISA method. Aused secondary enzyme-labeled antibody is horse radishperoxidase-labeled goat anti-rabbit IgC and the negative control is aPBS solution. Detection results are shown in FIG. 5. It can be seen fromthe results that, the antibody titer is extremely high and is greaterthan 1:1×10⁶.

Embodiment 9: Preparation of an Antigen Immunoassay Kit for Cryptococcusneoformans Capsular Polysaccharide

1. Preparation of an Enzyme-Labeled Carrier

{circle around (1)} diluting GXM to 25 ng/100 μL with a coating buffersolution to obtain a GXM coating solution, adding the GXM coatingsolution into wells of an ELISA plate, respectively adding 200 μL of thecoating solution into each well, and coating the ELISA plate at 2-8° C.for 8 h;

{circle around (2)} adding a blocking solution into the wells of theELISA plate, respectively adding 200 μL of the blocking solution intoeach well, and blocking at 37° C. for 30 min; and

{circle around (3)} removing the blocking solution, and standing at aconstant temperature of 37° C. for 30 min, thereby obtaining a GXMcoated enzyme-labeled carrier.

The coating buffer solution is 0.01 mol/L of PBS buffer solution with apH of 7.0-7.4.

The prepared blocking solution: 0.01 mol/L of PBS buffer solution with apH of 7.0-7.4 containing 3% of skim milk powder.

2. Preparation of a GXM Standard Substance

A GXM antigen is diluted into the concentrations of 100, 32, 10, 6.4 and3.2 ng/mL by using a sample diluent.

3. Preparation of an Anti-GXM Polyclonal Enzyme-Labeled AntibodySolution

An enzyme used for labeling is AP, and preparation of an anti-GXMpolyclonal enzyme-labeled antibody is performed by adopting aglutaraldehyde cross-linking method.

The AP-labeled anti-GXM polyclonal enzyme-labeled antibody is diluted byan AP conjugate stabilizer according to a ratio of 1:2000.

4. Preparation of a Sample Treatment Solution, a Wash Concentrate, aSample Diluent, a Substrate Solution and a Stop Solution

the sample treatment solution: dissolving ethylenediamine tetraaceticacid disodium salt with ultrapure water, and preparing into 0.05 mol/Lof EDTA solution, wherein a pH value is 4.0-4.8;

the wash concentrate: comprising the following components in parts byweight: 96.0 parts of sodium chloride, 2.40 parts of potassium chloride,42.96 parts of sodium phosphate dibasic dodecahydrate, 2.88 parts ofmonopotassium phosphate, 0.05 part of Tween-20 and 1000 parts ofultrapure water;

the sample diluent: artificial serum;

the substrate solution p-NPP solution;

the stop solution: dissolving 54.7 mL of concentrated sulfuric acid intohigh purity water and diluting to 100 mL, thereby obtaining 10 mol/L ofsulfuric acid solution.

5. The anti-GXM polyclonal enzyme-labeled antibody, the GXM standardsubstance, the sample treatment solution, the wash concentrate, thesample diluent, the substrate solution and the stop solution arerespectively filled into corresponding reagent bottles; the reagentbottles are fixed by a sponge carrier and are placed into a kit bodywith a GXM-coated enzyme-labeled antibody and a sealing film together.

Embodiment 10: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

In a GXM coating solution, the GXM is diluted to 25 ng/100 μL with 0.1mol/L of PBS buffer solution with a pH of 7.6-8.0, and the rest stepsare the same as those in Embodiment 9.

Embodiment 11: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

In a GXM coating solution, the GXM is diluted to 25 ng/100 μL with 0.2mol/L of PBS buffer solution with a pH of 7.6-8.0, and the rest stepsare the same as those in Embodiment 9.

Embodiment 12: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

In a GXM coating solution, the GXM is diluted to 25 ng/100 μL with 0.05mol/L of CBS buffer solution with a pH of 9.0-9.6, and the rest stepsare the same as those in Embodiment 9.

Embodiment 13: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

In a GXM coating solution, the GXM is diluted to 25 ng/100 μL with 0.1mol/L of CBS buffer solution with a pH of 9.0-9.6, and the rest stepsare the same as those in Embodiment 9.

Embodiment 14: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

In a GXM coating solution, the GXM is diluted to 25 ng/100 μL with 0.2mol/L of CBS buffer solution with a pH of 9.0-9.6, and the rest stepsare the same as those in Embodiment 9.

Embodiment 15: Preparation of an Antigen Immunoassay Kit for aCryptococcus neoformans Capsular Polysaccharide

In a GXM coating solution, the GXM is diluted to 25 ng/100 μL with 0.05mol/L of Tris buffer solution with a pH of 10.0-10.6, and the rest stepsare the same as those in Embodiment 9.

Embodiment 16: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

1. Preparation of an Enzyme-Labeled Carrier

{circle around (1)} diluting GXM to 500 ng/100 μL with a coating buffersolution to obtain a GXM coating solution, adding the GXM coatingsolution into wells of an ELISA plate, respectively adding 100 μL of thecoating solution into each well, and coating the ELISA plate at 2-8° C.for 12 h;

{circle around (2)} adding a blocking solution into the wells of theELISA plate, respectively adding 100 μL of the blocking solution intoeach well, and blocking at 37° C. for 60 min; and

{circle around (3)} removing the blocking solution, and standing at aconstant temperature of 37° C. for 60 min, thereby obtaining a GXMcoated enzyme-labeled carrier.

The coating buffer solution is 0.01 mol/L of PBS buffer solution with apH of 7.2-7.4.

The prepared blocking solution 0.01 mol/L of PBS buffer solution with apH of 7.2-7.4 containing 4% of skim milk powder.

2. Preparation of a GXM Standard Substance

A GXM antigen is diluted into the concentrations of 100, 32, 10, 6.4 and3.2 ng/mL by using a sample diluent.

3. Preparation of an Anti-GXM Polyclonal Enzyme-Labeled AntibodySolution

An enzyme used for labeling is HRP, and preparation of an anti-GXMpolyclonal enzyme-labeled antibody is performed by adopting a periodateoxidation method.

The HRP-labeled anti-GXM polyclonal enzyme-labeled antibody is dilutedby an HRP conjugate stabilizer according to a ratio of 1:10000.

4. Preparation of a Sample Treatment Solution, a Wash Concentrate, aSample Diluent, a Substrate Solution and a Stop Solution

the sample treatment solution: dissolving ethylenediamine tetraaceticacid disodium salt with ultrapure water, and preparing into 0.1 mol/L ofEDTA solution, wherein a pH value is 4.0-4.8;

the wash concentrate: comprising the following components in parts byweight: 96.0 parts of sodium chloride, 2.40 parts of potassium chloride,42.96 parts of sodium phosphate dibasic dodecahydrate, 2.88 parts ofmonopotassium phosphate, 0.05 part of Tween-20 and 1000 parts ofultrapure water;

the sample diluent: artificial cerebrospinal fluid;

the substrate solution: TMB solution;

the stop solution: diluting concentrated sulfuric acid and ultrapurewater according to a volume ratio of 1:8, thereby obtaining 2 mol/L ofsulfuric acid solution.

5. The anti-GXM polyclonal enzyme-labeled antibody, the GXM standardsubstance, the sample treatment solution, the wash concentrate, thesample diluent, the substrate solution and the stop solution arerespectively filled into corresponding reagent bottles; the reagentbottles are fixed by a sponge carrier and are placed into a kit bodywith a GXM-coated enzyme-labeled antibody and a sealing film together.

Embodiment 17: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

The blocking solution is 0.2 mol/L of PBS buffer solution with a pH of7.6-8.0 containing 5% of skim milk powder, and the rest steps are thesame as those in Embodiment 16.

Embodiment 18: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

The blocking solution is 0.2 mol/L of PBS buffer solution with a pH of7.6-8.0 containing 1% of BSA, and the rest steps are the same as thosein Embodiment 16.

Embodiment 19: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

The blocking solution is 0.2 mol/L of PBS buffer solution with a pH of7.6-8.0 containing 2% of BSA, and the rest steps are the same as thosein Embodiment 16.

Embodiment 20: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

The blocking solution is 0.2 mol/L of PBS buffer solution with a pH of7.6-8.0 containing 4% of BSA, and the rest steps are the same as thosein Embodiment 16.

Embodiment 21: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

1. Preparation of an Enzyme-Labeled Carrier

{circle around (1)} diluting GXM to 2 μg/100 μL with a coating buffersolution to obtain a GXM coating solution, adding the GXM coatingsolution into wells of an ELISA plate, respectively adding 60 μL of thecoating solution into each well, and coating the ELISA plate at 2-8° C.for 16 h;

{circle around (2)} adding a blocking solution into the wells of theELISA plate, respectively adding 60 μL of the blocking solution intoeach well, and blocking at 37° C. for 90 min; and

{circle around (3)} removing the blocking solution, and standing at aconstant temperature of 37° C. for 90 min, thereby obtaining a GXMcoated enzyme-labeled carrier.

The coating buffer solution is 0.1 mol/L of PBS buffer solution with apH of 7.4 (comprising the following components in parts by weight: 4.25parts of sodium chloride, 15.40 parts of sodium phosphate dibasicdodecahydrate, 0.95 part of monopotassium phosphate and 500 parts ofultrapure water).

The prepared blocking solution: 0.1 mol/L of PBS buffer solution with apH of 7.2-7.4 containing 4% of BSA.

2. Preparation of a GXM Standard Substance

A GXM antigen is diluted into the concentrations of 100, 32, 10, 6.4 and3.2 ng/mL by using a sample diluent.

3. Preparation of an Anti-GXM Polyclonal Enzyme-Labeled AntibodySolution

An enzyme used for labeling is HRP, and preparation of an anti-GXMpolyclonal enzyme-labeled antibody is performed by adopting a periodateoxidation method.

The HRP-labeled anti-GXM polyclonal enzyme-labeled antibody is dilutedby an HRP conjugate stabilizer according to a ratio of 1:20000.

4. Preparation of a Sample Treatment Solution, a Wash Concentrate, aSample Diluent, a Substrate Solution and a Stop Solution

the sample treatment solution: dissolving ethylenediamine tetraaceticacid disodium salt with ultrapure water, and preparing into a 0.2 mol/Lof EDTA solution, wherein a pH value is 4.0-4.8;

the wash concentrate: comprising the following components in parts byweight: 96.0 parts of sodium chloride, 2.40 parts of potassium chloride,42.96 parts of sodium phosphate dibasic dodecahydrate, 2.88 parts ofmonopotassium phosphate, 0.05 part of Tween-20 and 1000 parts ofultrapure water;

the sample diluent: artificial serum;

the substrate solution: TMB solution;

the stop solution: 98% of concentrated sulfuric acid.

5. The anti-GXM polyclonal enzyme-labeled antibody, the GXM standardsubstance, the sample treatment solution, the wash concentrate, thesample diluent, the substrate solution and the stop solution arerespectively filled into corresponding reagent bottles; the reagentbottles are fixed by a sponge carrier and are placed into a kit bodywith a GXM-coated enzyme-labeled antibody and a sealing film together.

Embodiment 22: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving sodium dodecyl sulfate with ultrapure water, preparing 0.01mol/L of SDS solution with a pH of 8.5-10.0, and performing the reststeps which are the same as those in Embodiment 21.

Embodiment 23: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving sodium dodecyl sulfate with ultrapure water, preparing 0.05mol/L of SDS solution with a pH of 8.5-10.0, and performing the reststeps which are the same as those in Embodiment 21.

Embodiment 24: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving sodium dodecyl sulfate with ultrapure water, preparing 0.1mol/L of SDS solution with a pH of 8.5-10.0, and performing the reststeps which are the same as those in Embodiment 21.

Embodiment 25: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving glycine with ultrapure water, preparing 0.07 mol/L of glycinesolution, regulating a pH to 2.2-2.8 with a concentrated HCL solution,and performing the rest steps which are the same as those in Embodiment21.

Embodiment 26: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving glycine with ultrapure water, preparing 0.13 mol/L of glycinesolution, regulating a pH to 2.2-2.8 with a concentrated HCL solution,and performing the rest steps which are the same as those in Embodiment21.

Embodiment 27: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving glycine with ultrapure water, preparing 0.2 mol/L of glycinesolution, regulating a pH to 2.2-2.8 with a concentrated HCL solution,and performing the rest steps which are the same as those in Embodiment21.

Embodiment 28: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving pronase with ultrapure water, preparing 0.05 mg/mL of pronasesolution. A pH value of the pronase solution is 8.0-9.0, and the reststeps are the same as those in Embodiment 21.

Embodiment 29: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving pronase with ultrapure water, preparing 5 mg/mL of pronasesolution. A pH value of the pronase solution is 8.0-9.0, and the reststeps are the same as those in Embodiment 21.

Embodiment 30: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving pronase with ultrapure water, preparing 15 mg/mL of pronasesolution. A pH value of the pronase solution is 8.0-9.0, and the reststeps are the same as those in Embodiment 21.

Embodiment 31: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving urea with ultrapure water, and preparing 1 mol/L of urea. ApH value of the urea is 7.2-8.0, and the rest steps are the same asthose in Embodiment 21.

Embodiment 32: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving urea with ultrapure water, and preparing 4 mol/L of urea. ApH value of the urea is 7.2-8.0, and the rest steps are the same asthose in Embodiment 21.

Embodiment 33: Preparation of an Antigen Immunoassay Kit forCryptococcus neoformans Capsular Polysaccharide

A preparation method of a sample treatment solution comprises the steps:dissolving urea with ultrapure water, and preparing 8 mol/L of urea. ApH value of the urea is 7.2-8.0, and the rest steps are the same asthose in Embodiment 21.

Embodiment 34: Operating Steps of a GXM Immunoassay Kit

1. Sample Treatment

1) mixing a to-be-detected sample and a sample treatment solutionaccording to a volume ratio of 1:1, and adding the mixture into aboiling water bath for 1 min;

2) centrifuging 1000 g of mixed solution after the water bath for 1 min;and

3) detecting by using a supernatant after centrifuging.

2. Detection Steps

1) taking the antigen immunoassay kit for the Cryptococcus neoformanscapsular polysaccharide prepared in Embodiments 9-15, and taking out anantigen-coated enzyme-labeled carrier;

2) preparing a working washing solution: diluting a wash concentrate by20 times (adding 19 parts of sterile deionized water or ultrapure waterinto 1 part of the wash concentrate);

3) sample mixing: respectively setting a standard curve group and ato-be-detected sample group, wherein

the standard curve group: each standard curve paint (3.2, 6.4, 10, 32and 100 ng/mL)

the to-be-detected sample group: to-be-detected samples treated in thestep 1 are repeatedly detected by 10 times;

respectively performing equivalent volume mixing on the two groups and aGXM enzyme-labeled antibody, and incubating at 37° C. for 20 min;

4) sample transfer: transferring a mixed solution in the step 3) intoELISA plate wells, adding 60 μL of the mixed solution into each well,and incubating at 37° C. for 20 min;

5) washing: removing liquid from the ELISA plate wells, adding not lessthan 300 μL of the working washing solution into each well each time,standing for 40 s, patting to be dry, repeating the above washingoperation, and totally washing for 5 times;

6) developing: adding 60 μL of a substrate solution p-NPP into each wellafter washing, incubating at 37° C. for 15 min, and keeping out of thesun;

7) stopping: adding 50 μL of stop solution into each well, mixing to beuniform, and reading an absorbance value at 405 nm; and

8) result judgment: respectively inputting determined absorbance valuesof the standard solution and the to-be-detected samples into a computer,calculating concentration values of the GXM in each to-be-detectedsample according to semi-logarithmic standard curves and equations drawnby calculation software and the absorbance values of the to-be-detectedsamples, calculating CV values, and comparing repeatability of samplesdetected by each kit.

TABLE 6 Sample detection results of kits prepared in Embodiments 9-15Detection Embodiment Embodiment Embodiment Embodiment EmbodimentEmbodiment Embodiment kit 9 10 11 12 13 14 15 Antigen 3.2 1.208 1.3291.170 1.244 1.276 1.160 1.276 concentration 6.4 1.169 1.285 1.131 1.2041.234 1.122 1.235 of 10 1.083 1.191 1.048 1.115 1.143 1.040 1.144standard 32 0.776 0.853 0.751 0.799 0.819 0.745 0.819 curve 100 0.4060.447 0.393 0.418 0.429 0.390 0.429 (μg/L) To-be-detected OD₄₅₀ 0.8640.948 0.819 0.894 0.904 0.797 0.903 sample 0.869 0.931 0.825 0.878 0.9080.847 0.905 0.865 0.939 0.809 0.870 0.934 0.817 0.896 0.852 0.942 0.8350.890 0.901 0.833 0.926 0.817 0.942 0.812 0.874 0.910 0.794 0.865 0.8970.949 0.827 0.929 0.891 0.822 0.903 0.844 0.932 0.860 0.884 0.911 0.8240.892 0.879 0.954 0.829 0.912 0.894 0.814 0.904 0.854 0.932 0.832 0.8950.916 0.811 0.897 0.852 0.924 0.814 0.893 0.938 0.824 0.916 Antigen24.57 24.69 25.90 24.22 25.12 27.24 25.22 concentration 24.17 25.8325.45 25.38 24.84 23.18 25.08 (μg/L) 24.48 25.28 26.77 26.00 22.95 25.5225.75 25.48 25.07 24.61 24.51 25.31 24.28 23.55 28.32 25.05 26.49 25.6924.67 27.48 28.11 22.13 24.62 25.29 21.73 26.09 25.16 25.24 26.06 25.7622.66 24.93 24.60 24.96 26.05 23.41 24.25 25.09 22.96 25.88 25.83 25.1225.34 25.81 24.87 24.13 24.23 26.05 25.68 25.46 26.36 26.28 24.26 22.7025.00 24.26 CV % 6.6% 2.6% 4.7% 5.3% 4.5% 5.0% 4.7%

Detection results of the kits prepared in Embodiments 9-15 for the samesample are shown in Table 4. It can be seen from data in Table 6 that,the CV value of each kit for the sample detection results is less than7%, which indicates that the dispersion degree of each kit for thesample detection results is low and repeatability is good so the kit canbe used in immunodetection of the Cryptococcus neoformans capsularpolysaccharide antigen; and moreover, the CV value detected by the kitprepared in Embodiment 10 is the lowest, which is 2.6%, which indicatesthat the kit has the best detection repeatability in the test of thepresent embodiment.

Embodiment 35: Operating Steps of a GXM Immunoassay Kit

1. Sample Treatment

1) mixing a to-be-detected sample and a sample treatment solutionaccording to a volume ratio of 3:1, and adding the mixture into aboiling water bath for 5 min;

2) centrifuging 5000 g of mixed solution after the water bath for 5 min;and

3) detecting by using a supernatant after centrifuging.

2. Detection Steps

1) taking the antigen immunoassay kit for the Cryptococcus neoformanscapsular polysaccharide prepared in Embodiments 16-20, and taking out anantigen-coated enzyme-labeled carrier;

2) preparing a working washing solution: diluting a wash concentrate by20 times (adding 19 parts of sterile deionized water or ultrapure waterinto 1 part of the wash concentrate);

3) sample mixing: respectively setting a standard curve group and ato-be-detected sample group, wherein

the standard curve group: each standard curve point (3.2, 6.4, 10, 32and 100 ng/mL)

the to-be-detected sample group: to-be-detected samples treated arerepeatedly detected by 10 times;

respectively performing equivalent volume mixing on the two groups and aGXM enzyme-labeled antibody, and incubating at 37° C. for 40 min;

4) sample transfer: transferring a mixed solution in the step 3) intoELISA plate wells, adding 80 μL of the mixed solution into each well,and incubating at 37° C. for 40 min;

5) washing: removing liquid in the ELISA plate wells, adding not lessthan 300 μL of the working washing solution into each well each time,standing for 40 s, patting dry, repeating the above washing operation,and totally washing for 5 times;

6) developing: adding 80 μL of substrate solution into each well afterwashing, incubating at 37° C. for 15 min, and keeping out of the sun;

7) stopping: adding 50 μL of a stop solution into each well, mixing tobe uniform, and reading an absorbance value at 450 nm; and

8) result judgment: respectively inputting determined absorbance valuesof the standard solution and the to-be-detected samples into a computer,calculating concentration values of the GXM in each to-be-detectedsample according to semi-logarithmic standard curves and equations drawnby calculation software and the absorbance values of the to-be-detectedsamples, calculating CV values, and comparing repeatability of samplesdetected by each kit.

TABLE 7 Sample detection results of kits prepared in Embodiments 16-20Detection Embodiment Embodiment Embodiment Embodiment Embodiment kit 1617 18 19 20 Antigen 3.2 1.206 1.326 1.167 1.242 1.273 concentration 6.41.166 1.283 1.129 1.201 1.232 of 10 1.081 1.189 1.046 1.113 1.141standard 32 0.774 0.852 0.750 0.797 0.817 curve 100 0.405 0.446 0.3920.417 0.428 (μg/L) To-be-detected OD₄₅₀ 0.852 0.948 0.818 0.882 0.910sample 0.851 0.959 0.856 0.903 0.910 0.843 0.939 0.826 0.870 0.895 0.8630.987 0.835 0.894 0.908 0.854 0.917 0.836 0.887 0.910 0.867 0.950 0.8050.871 0.899 0.852 0.927 0.825 0.852 0.909 0.857 0.932 0.832 0.872 0.9130.846 0.923 0.831 0.890 0.891 0.859 0.940 0.813 0.891 0.936 Antigen25.31 24.56 25.90 24.98 24.54 concentration 25.36 23.80 22.90 23.4324.50 (μg/L) 26.03 25.23 25.25 25.89 25.62 24.44 21.95 24.52 24.06 24.6725.17 26.77 24.45 24.56 24.54 24.16 24.42 27.00 25.78 25.36 25.33 26.0825.32 27.29 24.60 24.90 25.68 24.80 25.71 24.30 25.76 26.37 24.87 24.3525.90 24.74 25.15 26.28 24.26 22.70 CV % 2.3% 5.7% 4.5% 4.5% 3.6%

Detection results of the kits prepared in Embodiments 16-20 for the samesample are shown in Table 7. It can be seen from data in Table 7 that,the CV value of each kit for the sample detection results is less than6%, which indicates that the dispersion degree of each kit for thesample detection results is low and repeatability is good so the kit canbe used in immunodetection of the Cryptococcus neoformans capsularpolysaccharide antigen; and moreover, the CV value detected by the kitprepared in Embodiment 16 is the lowest, which is 2.3%, which indicatesthat the kit has the best detection repeatability in the test of thepresent embodiment.

Embodiment 36: Operating Steps of a GXM Immunoassay Kit

1. Sample Treatment

1) mixing a to-be-detected sample and a sample treatment solutionaccording to a volume ratio of 5:1, and adding the mixture into aboiling water bath for 10 min;

2) centrifuging 10000 g of a mixed solution after the water bath for 10min; and

3) detecting by using a supernatant after centrifuging.

2. Detection Steps

1) taking the antigen immunoassay kit for the Cryptococcus neoformanscapsular polysaccharide prepared in Embodiments 21-33, and taking out anantigen-coated enzyme-labeled carrier;

2) preparing a working washing solution: diluting a wash concentrate by20 times (adding 19 parts of sterile deionized water or ultrapure waterinto 1 part of the wash concentrate);

3) sample mixing: respectively setting a standard curve group and ato-be-detected sample group, wherein

the standard curve group: each standard curve point (3.2, 6.4, 10, 32and 100 ng/mL)

the to-be-detected sample group: to-be-detected samples treated arerepeatedly detected by 10 times;

respectively performing equivalent volume mixing on the two groups and aGXM enzyme-labeled antibody, and incubating at 37° C. for 60 min;

4) sample transfer: transferring a mixed solution in the step 3) intoELISA plate wells, adding 100 μL of the mixed solution into each well,and incubating at 37° C. for 60 min;

5) washing: removing liquid from the ELISA plate wells, adding not lessthan 300 μL of the working washing solution into each well each time,standing for 40 s, patting to be dry, repeating the above washingoperation, and totally washing for 5 times;

6) developing: adding 100 μL of a substrate solution into each wellafter washing, incubating at 37° C. for 15 min, and keeping out of thesun;

7) stopping: adding 50 μL of stop solution into each well, mixing to beuniform, and reading an absorbance value at 450 nm; and

8) result judgment: respectively inputting determined absorbance valuesof the standard solution and the to-be-detected samples into a computer,calculating concentration values of the GXM in each to-be-detectedsample according to semi-logarithmic standard curves and equations drawnby calculation software and the absorbance values of the to-be-detectedsamples, calculating CV values, and comparing repeatability of samplesdetected by each kit.

TABLE 8-1 Sample detection results of kits prepared in Embodiments 21-27Detection Embodiment Embodiment Embodiment Embodiment EmbodimentEmbodiment Embodiment kit 21 22 23 24 25 26 27 Antigen 3.2 1.242 1.3661.202 1.279 1.311 1.244 1.369 concentration 6.4 1.201 1.321 1.163 1.2371.268 1.204 1.324 of 10 1.113 1.224 1.078 1.146 1.175 1.115 1.227standard 32 0.797 0.877 0.772 0.821 0.842 0.799 0.879 curve 100 0.4170.459 0.404 0.43 0.441 0.418 0.46 (μg/L) To-be-detected OD₄₅₀ 0.8610.968 0.856 0.889 0.937 0.885 0.952 sample 0.879 0.929 0.864 0.91 0.9380.893 0.982 0.882 0.955 0.863 0.911 0.922 0.873 0.958 0.867 0.974 0.8240.922 0.936 0.895 1.019 0.89 0.981 0.867 0.924 0.942 0.863 0.971 0.8630.961 0.857 0.905 0.902 0.879 0.956 0.879 0.981 0.841 0.916 0.947 0.8830.971 0.883 0.951 0.857 0.914 0.914 0.883 0.955 0.875 0.978 0.858 0.9010.93 0.884 0.98 0.915 0.963 0.838 0.888 0.964 0.885 0.958 Antigen 26.5925.13 24.84 26.42 24.54 24.88 26.41 concentration 25.14 27.87 24.2324.83 24.5 24.33 24.3 (μg/L) 24.98 26 24.27 24.73 25.62 25.8 25.93 26.0624.7 27.38 23.95 24.67 24.14 21.91 24.39 24.27 23.96 23.84 24.19 26.5825.04 26.38 25.59 24.77 25.21 27.11 25.31 26.1 25.21 24.28 26 24.4 23.925.07 25.03 24.9 26.3 24.76 24.57 26.25 25.02 26.14 25.49 24.46 24.725.52 25.09 24.95 24.43 22.59 25.47 26.24 26.47 22.7 24.89 25.98 CV %4.5% 4.4% 4.3% 3.7% 5.0% 2.8% 5.4%

TABLE 8-2 Sample detection results of kits prepared in Embodiments 28-33Deletion Embodiment Embodiment Embodiment Embodiment EmbodimentEmbodiment kit 28 29 30 31 32 33 Antigen 3.2 1.205 1.282 1.314 1.1951.314 1.407 concentration 6.4 1.165 1.24 1.271 1.156 1.272 1.361 of 101.08 1.149 1.178 1.071 1.178 1.261 standard 32 0.774 0.823 0.844 0.7670.844 0.903 curve 100 0.405 0.431 0.442 0.402 0.442 0.473 (μg/L)To-be-detected OD₄₅₀ 0.856 0.902 0.929 0.849 0.909 0.988 sample 0.8540.905 0.939 0.849 0.936 0.984 0.859 0.922 0.901 0.835 0.96 0.978 0.870.913 0.965 0.819 0.972 1 0.841 0.912 0.956 0.856 0.92 0.985 0.867 0.8950.926 0.859 0.937 1.001 0.855 0.896 0.921 0.866 0.942 1.015 0.838 0.9010.958 0.826 0.949 0.977 0.839 0.935 0.959 0.85 0.925 0.999 0.845 0.9250.915 0.829 0.95 0.998 Antigen 24.99 25.62 25.28 24.95 26.76 25.7concentration 25.17 25.38 24.56 24.92 24.82 25.99 (μg/L) 24.71 24.1127.38 26.04 23.15 26.38 23.92 24.81 22.79 27.32 22.36 24.89 26.19 24.8323.39 24.38 25.93 25.9 24.1 26.08 25.49 24.15 24.76 24.82 25.02 26.0325.87 23.64 24.4 23.94 26.37 25.64 23.31 26.76 23.93 26.44 26.32 23.223.24 24.89 25.58 24.98 25.86 23.95 26.33 26.49 23.84 25.01 CV % 3.5%3.9% 6.3% 4.8% 5.4% 3.2%

Detection results of the kits prepared in Embodiments 21-33 for the samesample are shown in Table 8. It can be seen from data in Table 8 that,the CV value of each kit for the sample detection results is less than7%, which indicates that the dispersion degree of each kit for thesample detection results is low and repeatability is good so the kit canbe used in immunodetection of the Cryptococcus neoformans capsularpolysaccharide antigen; and moreover, the CV value detected by the kitprepared in Embodiment 26 is the lowest, which is 2.8%, which indicatesthat the kit has the best detection repeatability in the test of thepresent embodiment.

Embodiment 37: A Clinical Application of a GXM Immunoassay Kit

1. Drawing a Standard Curve

steps: taking a kit prepared in Embodiment 16, obtaining a measuredvalue of each of standard curve points (3.2, 6.4, 10, 32 and 100 ng/mL)shown in Table 9 according to steps in Embodiment 35, and making astandard curve shown in FIG. 6 by utilizing data in Table 9 and taking alogarithmic value of concentrations of a GXM antigen in a sample as ahorizontal axis (x axis) and taking an absorbance value measured at 450nm as a vertical axis (y axis), thereby obtaining a standard curveequation:y=A+(B−A)/[1+e{circumflex over ( )}(−a*x+b)]

wherein A=1.24414;

-   -   B=0.14362;    -   a=2.92462;    -   b=4.66878.        Linear correlation R{circumflex over ( )}2=0.99946, and the        standard curve equation is fitted well.

2. Determination of a Reference Value of the GXM Immunoassay Kit

steps: taking 30 positive samples which are clinically diagnosed to beinfected with cryptococcus, taking 200 normal human samples,centrifuging the samples, diluting and performing other treatments,determining OD₄₅₀ value according to steps in Embodiment 30 by utilizingthe kit prepared in Embodiment 11, and calculating antigen concentrationvalues shown in Table 10 according to the standard curve equation andabsorbance values of the samples;

calculating and detecting antigen concentrations according to results ofthe standard curve; by detecting the 200 normal human samples, taking anantigen concentration value in a 95% confidence interval as a Cut-offupper limit: x (mean)+2 s(standard deviation)=5.46+2*2.3=10; bydetecting 30 positive patients, taking an antigen concentration value inthe 95% confidence interval as a Cut-off lower limit: x (mean)−2s(standard deviation)=23.15−2*8.57=6; and determining a patient with theantigen concentration value of 6 ng/ml-10 ng/ml as a suspected patient,thereby obtaining a judgment standard reference value of the GXMimmunoassay kit shown in Table 11.

TABLE 9 Detected standard curve Antigen concentration of standardsubstance (ng/mL) OD₄₅₀ 3.2 1.196 6.4 1.157 10 1.072 32 0.768 100 0.402

TABLE 10 ELISA clinical detection results of the GXM immunoassay kit GXMconcentration Groups Cases x ± s(ng/mL) Positive rate (%) Normal person200 5.46 ± 2.3  3.5 (7/200) Positive patient 30 23.15 ± 8.57 93* (28/30)Note: *represents that P < 0.01 compared with normal persons.

TABLE 11 Judgment standard reference value of the GXM immunoassay kitPositive Suspected Negative Antigen concentration 6 ng/mL ≤ antigenAntigen concentration ≥10 ng/mL concentration ≤10 ng/mL ≤6 ng/mL

If the sample detection result is in the suspected interval, seconddetection shall be performed to determine the result.

Embodiment 38: Methodology Examination of a GXM Immunoassay Kit

1. Sensitivity Experiment

steps: taking a kit prepared in Embodiment 19, and detecting 20clinically diagnosed samples according to steps in Embodiment 34.

Diagnostic sensitivity is equal to a detected case number of positivesamples/a total case number of the positive samples×100%. Experimentalresults are shown in Table 12. It can be seen from data in Table 12that, the sensitivity of the GXM immunoassay kit used in the presentexperiment is higher than 85%.

TABLE 12 Experimental resuts of sensitivity detection Calculated antigenconcentration Judged No. OD₄₅₀ (μg/L) Result 1 0.053 1684.51 positive 20.061 1290.67 positive 3 0.09 701.97 positive 4 0.135 408.85 positive 50.227 213.34 positive 6 0.418 95.07 positive 7 0.319 137.96 positive 81.007 13.15 positive 9 0.103 582.55 positive 10 0.164 319.93 positive 110.313 141.46 positive 12 0.063 1219.78 positive 13 0.342 125.72 positive14 0.533 65.41 positive 15 1.122 6.56 suspected 16 0.062 1254.21positive 17 1.013 12.78 positive 18 1.049 10.62 suspected 19 0.126446.65 positive 20 1.08 8.85 suspected

2. Specificity Experiment

steps: taking a kit prepared in Embodiment 19, and detecting 20 healthyhuman samples according to steps in Embodiment 35.

Specificity is equal to a detected case number of negative samples/atotal case number of the negative samples×100%. Experimental results areshown in Table 13. It can be seen from data in Table 13 that, thesensitivity of the GXM immunoassay kit used in the present experiment ishigher than 90%.

TABLE 13 Experimental results of specificity detection Calculatedantigen No. OD₄₅₀ concentration (μg/L) Judged Result 1 1.391 Lower thanlower Negative detection limit 2 1.253 Lower than lower Negativedetection limit 3 1.211 1.99 Negative 4 1.328 Lower than lower Negativedetection limit 5 1.346 Lower than lower Negative detection limit 61.320 Lower than lower Negative detection limit 7 1.125 6.40 Suspected 81.309 Lower than lower Negative detection limit 9 1.289 Lower than lowerNegative detection limit 10 1.318 Lower than lower Negative detectionlimit 11 1.269 Lower than lower Negative detection limit 12 1.303 Lowerthan lower Negative detection limit 13 1.247 0.09 Negative 14 1.296Lower than lower Negative detection limit 15 1.474 Lower than lowerNegative detection limit 16 1.304 Lower than lower Negative detectionlimit 17 1.267 Lower than lower Negative detection limit 18 1.350 Lowerthan lower Negative detection limit 19 1.270 Lower than lower Negativedetection limit 20 1.330 Lower than lower Negative detection limit

3. Recovery Rate Experiment

steps: selecting normal human blood, respectively adding 90 μg/L and 30μg/L of Cryptococcus neoformans capsular polysaccharide antigen,detecting an antigen concentration according to steps in Embodiment 35by utilizing the kit prepared in Embodiment 19, and calculating a ratiobetween a true value and an expected value, thereby obtaining therecovery rate. The kit is qualified if the recovery rate is between 80%and 120%. Experimental results are shown in Table 14. It can be seenfrom data in Table 14 that, the recovery rate of the GXM immunoassay kitused in the present experiment is between 80% and 120%, and the recoveryrate is good.

TABLE 14 Experimental results of the recovery rate Calculated antigenRecovery OD₄₅₀ concentration (μg/L) rate Concentration of 0.419 105 114%added antigen is 0.475 90 97% 90 μg/L 0.447 100 104% Concentration of0.902 25 84% added antigen is 0.895 28 90% 30 μg/L 0.899 27 87%

4. Repeatability Experiment

1) Between-Run Precision

An acceptable quality level: testing the same sample once per day for 10consecutive workdays according to steps in Embodiment 35 by utilizingthe kits prepared in Embodiments 18 and 19, calculating a mean value M,a standard deviation SD and a variable coefficient CV, wherein thesample is qualified if the variable coefficient CV is less than or equalto 25%. Experimental results are shown in Table 15. It can be concludedfrom data in Table 15 that, the between-run precision (that is, thevariable coefficient CV) of the GXM immunoassay kit provided in thepresent invention is 15%, is less than 25% and conforms to the standard.

TABLE 15 Expermental results of the between-run precision Summarizedbetween-run precision Calculated antigen concentration Workday (d)(μg/L) 1 30.11 2 29.09 3 22.80 4 19.01 5 29.63 6 27.39 7 20.81 8 23.82 927.41 10  26.49 M 25.66 SD 3.85 CV 15%

2) Within-Run Precision

An acceptable quality level: testing 10 groups of data of the samesample in parallel in the same batch of experiments; determining twiceeach time; taking a mean value; calculating a corresponding antigenconcentration; and calculating a mean value M, a standard deviation SDand a variable coefficient CV, wherein the sample is qualified if thevariable coefficient CV is less than or equal to 15%. The within-runprecision (that is, the variable coefficient CV) of the GXM immunoassaykit provided in the present invention is 7%, is less than 15% andconforms to the standard.

TABLE 16 Experimental results of the within-run precision Calculatedantign concentration No. OD₄₅₀-1 OD₄₅₀-2 OD₄₅₀ (μg/L) 1 0.792 0.807 0.828.87 2 0.769 0.780 0.775 31.27 3 0.839 0.856 0.848 24.62 4 0.813 0.8190.816 27.40 5 0.794 0.822 0.808 28.13 6 0.802 0.822 0.812 27.76 7 0.8160.863 0.84 25.29 8 0.781 0.812 0.797 29.15 9 0.817 0.828 0.823 26.77 100.770 0.798 0.784 30.39 M 28.0 SD 2.1 CV 7%

5. Stability Experiment

steps: placing the GXM immunoassay kit prepared in Embodiment 18 in anenvironment of 37° C.; and detecting a standard quality control solution(55 μg/L) with a known concentration through a standard curve per dayfor consecutive 5 days, wherein if variation of the detected value (thatis, a variable coefficient CV) is less than 20%, the kit is proved to bestable. Experimental results are shown in Table 17. It can be seen fromdata in Table 17 that, the variable coefficient CV of the GXMimmunoassay kit prepared in Embodiment 8 adopted in the presentexperiment within 5 days is less than or equal to 20%, which indicatesthat the GXM immunoassay kit provided in the present invention is goodin stability.

TABLE 17 Experimental results of the stability 1 2 3 4 5 CV Blank 0.0560.060 0.060 0.050 0.061 Antigen 3.2 1.176 1.216 1.121 1.201 1.198concentration 6.4 1.156 1.157 1.108 1.099 1.108 of 10 1.037 1.107 1.0861.084 1.079 standard 32 0.771 0.764 0.767 0.752 0.772 curve 100 0.4030.4 0.399 0.391 0.402 (μg/L) Standard OD₄₅₀ 0.587 0.598 0.576 0.5810.563 quality Antigen 55.447 50.297 48.989 53.497 60.380 8% controlconcentration point (μg/L)

What is claimed is:
 1. A preparation method of Cryptococcus neoformanscapsular polysaccharide glucuronoxylomannan (GXM), comprising:performing crude extraction on Cryptococcus neoformans capsularpolysaccharide GXM; preparing an immunoaffinity chromatography columnfor the Cryptococcus neoformans capsular polysaccharide GXM; andpurifying a crude extract of the Cryptococcus neoformans capsularpolysaccharide GXM by utilizing the GXM immunoaffinity chromatographycolumn; and, the immunoaffinity chromatography column is preparedthrough following steps: (1) dissolving a monoclonal antibody ofCryptococcus neoformans capsular polysaccharide GXM in a solution, andmixing the monoclonal antibody with an affinity chromatography substrateto form thin homogenate; (2) washing the homogenate obtained in the step(1) with a cross-linking buffer solution, and centrifuging to obtain amixture of the antibody and the affinity chromatography substrate; (3)resuspending the mixture of the antibody and the affinity chromatographysubstrate obtained in step (2) with the cross-linking buffer solution,adding a difunctional binding agent into the obtained suspension,incubating, uniformly mixing, and separating the liquid from the solidto obtain an affinity chromatography substrate-antibody conjugatedcomplex; (4) washing the affinity chromatography substrate-antibodyconjugated complex obtained in the step (3) with a blocking solution;(5) resuspending the affinity chromatography substrate-antibodyconjugated complex obtained in the step (4) in the blocking solution,incubating, and uniformly mixing; and (6) packing: filling the affinitychromatography substrate-antibody conjugated complex obtained in thestep (5) into a chromatographic column, thereby obtaining theimmunoaffinity chromatography column for the Cryptococcus neoformanscapsular polysaccharide GXM; wherein, the affinity chromatographysubstrate-antibody conjugated complex obtained in step (5) is subjectedto a preservative treatment and then filled into the chromatographycolumn, and the preservative treatment comprises following steps:washing the affinity chromatography substrate-antibody conjugatedcomplex obtained in the step (5) with a PBS buffer solution,resuspending the complex in the PBS buffer solution, and addingmerthiolate for preservation.
 2. The preparation method according toclaim 1, wherein the extraction of the Cryptococcus neoformans capsularpolysaccharide GXM comprises the following steps: (1) culturingCryptococcus neoformans until a bacteria concentration reaches a laterlogarithmic phase; (2) performing autoclaved sterilization on bacteriumliquid, centrifuging to remove thalli, and reserving supernatant; (3)adding calcium acetate powder into the supernatant while stirring untilthe final concentration of the supernatant is 2-8%, and adding glacialacetic acid to regulate the pH value to be 4.6-5.2; and (4) addingethanol with a concentration of 90-98% into the solution obtained in thestep (3), standing overnight at 4° C., centrifuging, removing thesupernatant, drying and precipitating, thereby obtaining the crudeextract of the Cryptococcus neoformans capsular polysaccharide GXM. 3.The preparation method according to claim 1, wherein the step ofpurifying the crude extract of the Cryptococcus neoformans capsularpolysaccharide GXM by utilizing the GXM immunoaffinity chromatographycolumn comprises the following steps: (1) rinsing the GXM immunoaffinitychromatography column by utilizing a buffer solution which is the sameas a to-be-purified sample solution; (2) enabling the to-be-purifiedsample solution to flow through the chromatography column treated in thestep (1); (3) washing the column with a binding buffer solution; (4)washing the column with a pre-elution buffer solution; (5) performingstepwise elution, continuously enabling an elution buffer solution toflow through the chromatography column, and collecting each component indifferent tubes; and (6) detecting the content of the GXM in each tube,and merging all the tubes with high concentrations.
 4. The preparationmethod according to claim 3, wherein flow velocity of the to-be-purifiedsample in the step (2) is 0.5-1.5 mL/h.
 5. The preparation methodaccording to claim 3, wherein the binding buffer solution in the step(3) is selected from one of a PBS buffer solution, a Tris-HCl buffersolution and an acetic acid-sodium acetate buffer solution, and the useamount of the binding buffer solution is 10-25 times of the column bedvolume; and/or the pre-elution buffer solution in the step (4) is acarbonate buffer solution, and the use amount of the pre-elution buffersolution is 10-25 times of the column bed volume; and/or the elutionbuffer solution in the step (5) is selected from 0.1M glycine buffersolution with a pH of 3.0, a citrate-phosphate buffer solution with a pHof 3.0, a citrate-sodium citrate buffer solution with a pH of 3.0 and anacetate-sodium acetate buffer solution with a pH of 3.0, and the useamount of the elution buffer solution is 0.4-0.8 of the column bedvolume.
 6. The preparation method according to claim 1, wherein theaffinity chromatography substrate in the step (1) is selected fromprotein A microbeads, protein G microbeads and active microbeads; and/orthe solution in the step (1) is selected from a carbonate buffersolution and an acetic acid-sodium acetate buffer solution; and/or aratio of the affinity chromatography substrate to the solution in thestep (1) is as follows: 0.5-2.0 mL of the affinity chromatographysubstrate is added into every 10 mL of the solution; and a ratio of theaffinity chromatography substrate to the antibody is as follows: every 1mL of the affinity chromatography substrate is bound to 1-4 mg of themonoclonal antibody.
 7. The preparation method according to claim 1,wherein the cross-linking buffer solution in the step (2) is a 0.1-0.3mol/L of sodium borate solution with a pH value of 8.0-9.5, and the useamount of the buffer solution is 5-15 times of the volume of theaffinity chromatography substrate; and/or the cross-linking buffersolution in the step (3) is a sodium borate solution with aconcentration of 0.1-0.3 mol/L and a pH value of 8.3-9.5, and the useamount is 5-15 times of the volume of the affinity chromatographysubstrate.
 8. The preparation method according to claim 1, wherein thedifunctional binding agent in the step (3) is selected from dimethylheptandilate, carbonyldimidazole, cyanogen bromide, hydroxysuccinimideand acetyl iodine, and the use amount is as follows: the finalconcentration of the binding agent in the suspension is 15-25 mmol/L. 9.The preparation method according to claim 1, wherein the blockingsolution in the steps (4) and (5) is selected from ethanol amine andaminoethane solutions.
 10. The preparation method according to claim 1,wherein, the preparing an immunoaffinity chromatography column furthercomprises: (7) preserving the affinity chromatography substrate-antibodyconjugated complex obtained in step (6) or packed immunoaffinitychromatography column for the Cryptococcus neoformans capsularpolysaccharide GXM obtained in step (6) under a condition of 4° C., andenabling a performance to be stable within 1 year.
 11. The preparationmethod according to claim 10, wherein, the preparing an immunoaffinitychromatography column further comprises a step (8) of regeneration:washing and regenerating the chromatography column preserved under thecondition of 4° C. in the step (7) or the used chromatography columnwith a buffer solution which has a solvent the same as a to-be-purifiedsample, in an amount of 10-25 times of the volume of the column bedbefore use.
 12. The preparation method according to claim 1, wherein, aconcentration of the merthiolate is 0.005-0.015%.